CN101984046A - Corynebacterium glutamicum capable of producing succinic acid with high yield - Google Patents
Corynebacterium glutamicum capable of producing succinic acid with high yield Download PDFInfo
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- CN101984046A CN101984046A CN201010578530.6A CN201010578530A CN101984046A CN 101984046 A CN101984046 A CN 101984046A CN 201010578530 A CN201010578530 A CN 201010578530A CN 101984046 A CN101984046 A CN 101984046A
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- corynebacterium glutamicum
- succsinic acid
- high yield
- aerobic fermentation
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- 241000186226 Corynebacterium glutamicum Species 0.000 title claims abstract description 27
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 title abstract description 17
- 239000001384 succinic acid Substances 0.000 title abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims abstract description 14
- 230000004151 fermentation Effects 0.000 claims abstract description 14
- 230000001580 bacterial effect Effects 0.000 claims abstract description 10
- 239000002253 acid Substances 0.000 claims description 37
- 238000010564 aerobic fermentation Methods 0.000 claims description 20
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 13
- 240000008042 Zea mays Species 0.000 claims description 10
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 10
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 10
- 235000005822 corn Nutrition 0.000 claims description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- 239000004202 carbamide Substances 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- XSQUKJJJFZCRTK-UHFFFAOYSA-N urea group Chemical group NC(=O)N XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 8
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- 229930003756 Vitamin B7 Natural products 0.000 claims description 6
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 235000011912 vitamin B7 Nutrition 0.000 claims description 6
- 239000011735 vitamin B7 Substances 0.000 claims description 6
- -1 vitriol Chemical class 0.000 claims description 6
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 5
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 4
- 102000013275 Somatomedins Human genes 0.000 claims description 4
- 229930003779 Vitamin B12 Natural products 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 235000019163 vitamin B12 Nutrition 0.000 claims description 4
- 239000011715 vitamin B12 Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 3
- 159000000007 calcium salts Chemical class 0.000 claims description 3
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 3
- 159000000003 magnesium salts Chemical class 0.000 claims description 3
- 150000002696 manganese Chemical class 0.000 claims description 3
- 150000003016 phosphoric acids Chemical class 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 150000003751 zinc Chemical class 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
- 238000009423 ventilation Methods 0.000 claims description 2
- 235000013343 vitamin Nutrition 0.000 claims description 2
- 229930003231 vitamin Natural products 0.000 claims description 2
- 239000011782 vitamin Substances 0.000 claims description 2
- 229940088594 vitamin Drugs 0.000 claims description 2
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims 1
- 239000006227 byproduct Substances 0.000 abstract description 10
- 241000894006 Bacteria Species 0.000 abstract description 8
- 239000000126 substance Substances 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 239000003054 catalyst Substances 0.000 abstract 1
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 239000010970 precious metal Substances 0.000 abstract 1
- 239000000047 product Substances 0.000 abstract 1
- 230000009897 systematic effect Effects 0.000 abstract 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 12
- 239000002609 medium Substances 0.000 description 10
- 239000004310 lactic acid Substances 0.000 description 6
- 235000014655 lactic acid Nutrition 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 230000031018 biological processes and functions Effects 0.000 description 4
- 238000007664 blowing Methods 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000948980 Actinobacillus succinogenes Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000722954 Anaerobiospirillum succiniciproducens Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000029538 [Mannheimia] succiniciproducens Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
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- 239000008272 agar Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
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- 238000010884 ion-beam technique Methods 0.000 description 1
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- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 229920002961 polybutylene succinate Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- 238000003786 synthesis reaction Methods 0.000 description 1
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- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
Images
Abstract
The invention discloses a corynebacterium glutamicum capable of producing succinic acid with high yield. The systematic nomenclature of the corynebacterium glutamicum is corynebacterium glutamicum SA001 and the corynebacterium glutamicum is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No.3991. The invention further discloses a method of producing the succinic acid by utilizing the corynebacterium glutamicum capable of producing the succinic acid with high yield. Compared with the chemical methods, the method disclosed by the invention has the advantages of low energy consumption and less pollution; and in the method, non-fossil resources are used as raw materials, and precious metals are not required to be used as catalysts, thus the cost is low, the process can be controlled easily and the yield is high. Compared with the original bacterium, the bacterial strain provided by the invention has the advantages of single fermentation product, less by-products and high yield.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of Corynebacterium glutamicum of high yield succsinic acid.
Background technology
Succsinic acid has another name called Succinic Acid, is a kind of common natural organic acids, as the end product of tricarboxylic acid cycle reducing end, extensively is present in human body, animals and plants and the microorganism, occupies critical role in biological metabolism.Succsinic acid not only can be used as additive and is applied to industries such as food, medicine, can also be used for synthesizing 1, bulk chemical and PBS Biodegradable materials such as (poly butylene succinates) such as 4-butyleneglycol, tetrahydrofuran (THF) as C4 hardware and software platform compound simultaneously.
The preparation method of succsinic acid mainly comprises chemical synthesis and biological process.Compare chemical method and be difficult to control, problems such as productive rate is not high, purity is low, catalyzer costliness, it is with low cost that the acid of biological process ambroid has, the higher fixation of C O that has concurrently simultaneously of productive rate
2Characteristics, so biological process prepares the research focus that succsinic acid becomes this year.The main bacteria seed of biological process ambroid acid at present comprises: Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Escherichia coli, Mannheimia succiniciproducens etc.Wherein Corynebacterium glutamicum Corynebacteriumglutamicum is rapid because of its growth, culture condition is simple, tolerance is strong, fermentation condition is ripe, while is owing to the disappearance of part enzyme in its anaerobic metabolism approach, under anaerobic by product is less to make it, becomes the industrial strain of the production succsinic acid of tool potentiality.But wild Corynebacterium glutamicum is in the process of fermentative production succsinic acid, and invariably accompany by product L-lactic acid and acetate have influenced the quality of tunning, have increased the load of later separation.
Summary of the invention
Technical problem to be solved by this invention provides the Corynebacterium glutamicum that a plant height produces succsinic acid, does not produce by product L-lactic acid under its anaerobic condition.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The contriver is by the low energy ion beam implantttion technique, ATCC13032 carries out mutagenesis to Corynebacterium glutamicum (Corynebacterium glutamicum), obtain a strain Corynebacterium glutamicum through screening, but high yield succsinic acid under its anaerobism does not produce by product lactic acid simultaneously.This bacterial strain, its classification called after Corynebacterium glutamicum (Corynebacterium glutamicum) SA001, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) at present, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101, the numbering of registering on the books are CGMCC No.3991, and preservation date is on July 2nd, 2010.With this bacterium as producing bacterial strain.
CGMCC No.3991 bacterial strain has following character:
1, colonial morphology feature:
On nutrient agar, 30 ℃ streak culture is the moderates growth, the lawn wire, and 24h be white, is faint yellow behind the 48h slightly, increases slightly deeply with the prolongation color of incubation time.Surface wettability, smooth, glossy, no stickiness
2, physiology and biochemical characteristic:
A. culture temperature: 28～39 ℃, optimum temperuture is 30 ℃;
B. in pH 5.0～8.5 scopes, grow;
C. pigment production: do not produce pigment;
3, nutritional character:
Corynebacterium glutamicum can grow on synthetic medium, and medium component is clear and definite and cheap.This culture medium carbon source is generally glucose or corn saccharification liquid, and nitrogenous source can be selected Dried Corn Steep Liquor Powder, extractum carnis, peptone, yeast extract paste, urea and (NH for use
4)
2SO
4Deng, also comprise the inorganic salts that sylvite, sodium salt, calcium salt, magnesium salts, zinc salt, molysite, manganese salt, phosphoric acid salt, dihydrogen phosphate and hydrochloride etc. are commonly used in the substratum, decide according to different nitrogenous sources, need full the interpolation when being only nitrogen source with ammonium sulfate or urea, can be on a small quantity with Dried Corn Steep Liquor Powder, extractum carnis, peptone, yeast extract paste the time or do not add inorganic salt.
The method of utilizing the Corynebacterium glutamicum of above-mentioned high yield succsinic acid to produce succsinic acid, preserving number is the bacterial strain activation of CGMCCNo.3991 after, at 28～39 ℃ of following aerobic fermentation 12～16h, again at 28～39 ℃ of following anaerobically fermenting 12～36h;
Wherein, the aerobic fermentation substratum is a perfect medium: carbon source 10～40g/L, and nitrogenous source 10～20g/L, inorganic salt 0～3g/L, somatomedin 0～0.3mg/L, all the other are water, pH 5.0～7.5;
Wherein, in the anaerobic fermentation process, in the aerobic fermentation substratum, add carbon source 30～60g/L.
Wherein, described carbon source is glucose or corn saccharification liquid.
Wherein, described nitrogenous source is urea or Dried Corn Steep Liquor Powder, extractum carnis, peptone or yeast extract paste.
Wherein, described inorganic salt are any one or a few the combination in sylvite, manganese salt, molysite, magnesium salts, zinc salt, mantoquita, calcium salt, vitriol, phosphoric acid salt or the hydrochloride.
Wherein, described somatomedin is the combination of any one or two kinds in vitamin H and the VITAMIN.
The most preferred mode of production is: after preserving number is the bacterial strain activation of CGMCC No.3991, and at 30 ℃ of following aerobic fermentation 12h, aerobic stage ventilation 1v/vm, stirring 500rpm, again at 30 ℃ of following anaerobically fermenting 36h, anaerobic stages stirs 200rpm;
Wherein, described aerobic fermentation substratum is: glucose 20g/L, K
2HPO
43H
2O 1.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O 0.4g/L, FeSO
47H
2O 20mg/L, MnSO
4H
2O 20mg/L, urea 2.5g/L, vitamin H 100 μ g/L, vitamin B12 00 μ g/L, all the other are water, pH 7;
Wherein, in the anaerobic fermentation process, in the aerobic fermentation substratum, add carbon source 40g/L, at first begin and respectively add 20g/L during anaerobically fermenting 12h respectively at anaerobically fermenting.
Beneficial effect: the present invention compared with prior art has following advantage:
(1) compare with chemical method, energy consumption is low, pollutes for a short time, uses non-fossil resource as raw material, need not to use noble metal as catalyzer, and with low cost, process is easily controlled, the output height.
(2) compare with original bacterium, tunning is single, and by product is few, the output height.
Description of drawings
Fig. 1 detects collection of illustrative plates for the high performance liquid chromatography organic acid of bacterial strain CGMCC No.3991 anaerobically fermenting terminal point of the present invention.Wherein, the peak of label 1 is the response peak of succsinic acid.
Fig. 2 is that the high performance liquid chromatography organic acid of starting strain ATCC13032 anaerobically fermenting terminal point of the present invention detects collection of illustrative plates.Wherein, the peak of label 1 is the response peak of lactic acid, and 2 is the response peak of succsinic acid.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that embodiment is described only to be used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1:
Plate culture medium: peptone 10g/L, yeast extract paste 5g/L, NaCl 10g/L, agar 20g/L, pH7.0.
Aerobic fermentation substratum: glucose 20g/L, K
2HPO
43H
2O 1.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O0.4g/L, FeSO
47H
2O 20mg/L, MnSO
4H
2O 20mg/L, urea 2.5g/L, vitamin H 100 μ g/L, vitamin B12 00 μ g/L, initial pH7.0.5L fermentor tank liquid amount 3L sterilized 10 minutes for 115 ℃.
Be respectively that ATCC13032 and CGMCC No.3991 bacterium are at first in 30 ℃ of dull and stereotyped activated spawn with preserving number, connect the well-grown thalline of two rings behind the 24h and go into the 30mL fermention medium, 30 ℃, 200rpm cultivates 12h, transfers into the 3L fermention medium according to 3% (v/v) inoculum size then, blowing air 1v/vm, rotating speed 500rpm, 30 ℃, behind the aerobic fermentation 12h, change anaerobically fermenting over to, logical CO
20.2v/vm, rotating speed 200rpm, 30 ℃ of cultivations.Initially reach fermentation back 12h respectively at anaerobically fermenting and respectively add 20g/L glucose.Contain the 37.2g/L succsinic acid in the fermented liquid of CGMCC No. behind the fermentation 36h.The anaerobic stages glucose acid invert ratio reaches 93%, compares than ATCC13032, and succinic acid production has improved 325%, does not have by product lactic acid (seeing Fig. 1 and Fig. 2).
Embodiment 2:
Plate culture medium: with embodiment 1.
Aerobic fermentation substratum: corn saccharification liquid 400mL/L (sugar degree of corn saccharification liquid is about 50g/L), K
2HPO
43H
2O 1.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O 0.4g/L, FeSO
47H
2O 20mg/L, MnSO
4H
2O 20mg/L, urea 2.5g/L, vitamin H 100 μ g/L, vitamin B12 00 μ g/L, initial pH7.0.5L fermentor tank liquid amount 3L sterilized 10 minutes for 115 ℃.
Be respectively that ATCC13032 and CGMCC No.3991 bacterium are at first in 30 ℃ of dull and stereotyped activated spawn with preserving number, connect the well-grown thalline of two rings behind the 24h and go into the 30mL fermention medium, 30 ℃, 200rpm cultivates 12h, transfers into the 3L fermention medium according to 3% (v/v) inoculum size then, blowing air 1v/vm, rotating speed 500rpm, 30 ℃, behind the aerobic fermentation 14h, change anaerobically fermenting over to, logical CO
20.2v/vm, rotating speed 200rpm, 30 ℃ of cultivations.Initially reach fermentation back 12h respectively at anaerobically fermenting and respectively add 20g/L glucose.Contain the 36.2g/L succsinic acid in the fermented liquid of CGMCC No. behind the fermentation 36h.The anaerobic stages glucose acid invert ratio reaches 90.5%, compares than ATCC13032, and succinic acid production has improved 312%, does not have by product lactic acid
Embodiment 3:
Plate culture medium: with embodiment 1
Aerobic fermentation substratum: glucose 30g/L, peptone 5g/L, FeSO
47H
2O 20mg/L, MnSO
4H
2O20mg/L, initial pH7.0.5L fermentor tank liquid amount 3L sterilized 10 minutes for 115 ℃.
Be respectively that ATCC13032 and CGMCC No.3991 bacterium are at first in 30 ℃ of dull and stereotyped activated spawn with preserving number, connect the well-grown thalline of two rings behind the 24h and go into the 30mL fermention medium, 35 ℃, 200rpm cultivates 12h, transfers into the 3L fermention medium according to 3% (v/v) inoculum size then, blowing air 1v/vm, rotating speed 500rpm, 30 ℃, behind the aerobic fermentation 16h, change anaerobically fermenting over to, logical CO
20.2v/vm, rotating speed 200rpm, 35 ℃ of cultivations.Initially reach fermentation back 12h respectively at anaerobically fermenting and respectively add 20g/L glucose.Contain the 35.1g/L succsinic acid in the fermented liquid of CGMCC No. behind the fermentation 36h.The anaerobic stages glucose acid invert ratio reaches 87.75%, compares than ATCC13032, and succinic acid production has improved 298%, does not have by product lactic acid
Embodiment 4:
Plate culture medium: with embodiment 1
Aerobic fermentation substratum: glucose 20g/L, Dried Corn Steep Liquor Powder 10g/L, initial pH7.0.5L fermentor tank liquid amount 3L sterilized 10 minutes for 115 ℃.
Be respectively that ATCC13032 and CGMCC No.3991 bacterium are at first in 30 ℃ of dull and stereotyped activated spawn with preserving number, connect the well-grown thalline of two rings behind the 24h and go into the 30mL fermention medium, 30 ℃, 200rpm cultivates 12h, transfers into the 3L fermention medium according to 3% (v/v) inoculum size then, blowing air 1v/vm, rotating speed 500rpm, 30 ℃, behind the aerobic fermentation 16h, change anaerobically fermenting over to, logical CO
20.2v/vm, rotating speed 200rpm, 33 ℃ of cultivations.Initially reach fermentation back 12h respectively at anaerobically fermenting and respectively add 20g/L glucose.Contain the 35.8g/L succsinic acid in the fermented liquid of CGMCC No. behind the fermentation 36h.The anaerobic stages glucose acid invert ratio reaches 89.5%, compares than ATCC13032, and succinic acid production has improved 308%, does not have by product lactic acid.
Claims (7)
1. the Corynebacterium glutamicum of a high yield succsinic acid, its classification called after Corynebacterium glutamicum (Corynebacterium glutamicum) SA001, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, its deposit number is CGMCC No.3991.
2. utilize the method for the Corynebacterium glutamicum production succsinic acid of the described high yield succsinic acid of claim 1, after it is characterized in that preserving number is the bacterial strain activation of CGMCC No.3991, at 28～39 ℃ of following aerobic fermentation 12～16h, again at 28～39 ℃ of following anaerobically fermenting 12～36h;
Wherein, the aerobic fermentation substratum is a perfect medium: carbon source 10～40g/L, and nitrogenous source 10～20g/L, inorganic salt 0～3g/L, somatomedin 0～0.3mg/L, all the other are water, pH 5.0～7.5;
Wherein, in the anaerobic fermentation process, in the aerobic fermentation substratum, add carbon source 30～60g/L.
3. the Corynebacterium glutamicum of high yield succsinic acid according to claim 2 produces the method for succsinic acid, it is characterized in that described carbon source is glucose or corn saccharification liquid.
4. the Corynebacterium glutamicum of high yield succsinic acid according to claim 2 produces the method for succsinic acid, it is characterized in that described nitrogenous source is urea, ammonium sulfate, Dried Corn Steep Liquor Powder or yeast powder.
5. the Corynebacterium glutamicum of high yield succsinic acid according to claim 2 produces the method for succsinic acid, it is characterized in that described inorganic salt are any one or a few the combination in sylvite, manganese salt, molysite, magnesium salts, zinc salt, mantoquita, calcium salt, vitriol, phosphoric acid salt or the hydrochloride.
6. the Corynebacterium glutamicum of high yield succsinic acid according to claim 2 produces the method for succsinic acid, it is characterized in that described somatomedin is the combination of any one or two kinds in vitamin H and the VITAMIN.
7. the Corynebacterium glutamicum of high yield succsinic acid according to claim 2 produces the method for succsinic acid, after it is characterized in that preserving number is the bacterial strain activation of CGMCC No.3991, at 30 ℃ of following aerobic fermentation 12h, aerobic stage ventilation 1v/vm, stirring 500rpm, at 30 ℃ of following anaerobically fermenting 36h, anaerobic stages stirs 200rpm again;
Wherein, described aerobic fermentation substratum is: glucose 20g/L, K
2HPO
43H
2O 1.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O 0.4g/L, FeSO
47H
2O 20mg/L, MnSO
4H
2O 20mg/L, urea 2.5g/L, vitamin H 100 μ g/L, vitamin B12 00 μ g/L, all the other are water, pH 7;
Wherein, in the anaerobic fermentation process, in the aerobic fermentation substratum, add carbon source 40g/L, at first begin and respectively add 20g/L during anaerobically fermenting 12h respectively at anaerobically fermenting.
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| WO2013067850A1 (en) * | 2011-11-11 | 2013-05-16 | 南京工业大学 | Chemically defined culture medium for fermentation to produce succinic acid and application thereof |
| CN103642854A (en) * | 2013-12-03 | 2014-03-19 | 南京工业大学 | Method for producing succinic acid from corynebacterium glutamicum through immobilized repeated batch fermentation |
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| CN103642854B (en) * | 2013-12-03 | 2015-07-15 | 南京工业大学 | Method for producing succinic acid from corynebacterium glutamicum through immobilized repeated batch fermentation |
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